Effects of focused ultrasound in a “clean” mouse model of ultrasonic neuromodulation
Hongsun Guo, Hossein Salahshoor, Di Wu, Sangjin Yoo, Tomokazu Sato, Doris Y. Tsao, Mikhail G. Shapiro
iScience 2023, 26, 108372 · 10.1016/j.isci.2023.108372
Abstract
Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3 +/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.
Abstract via europepmc.
Exposures
Exposure 1: 270 kHz continuous FUS to visual cortex (near Lambda)
Target: visual cortex — “0.5 mm anterior and 2.5 mm lateral of Lambda (visual cortex)”
Device: Sonic Concepts · Sonic Concepts · H-115 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 270 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓? |
| Sonication duration (s) | 0.5 | ✓✓✓⚑ |
| Free-field pressure (kPa) | 100, 500, 900swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Continuous-wave FUS with a 500 ms pulse duration (PD) delivered at 270 kHz (transducer fundamental) to a target 0.5 mm anterior and 2.5 mm lateral of Lambda; 50 blocks of trials per animal, one trial of each stimulus type (sham, US at each pressure, light flash) presented once per block in random order, with a 20 s inter-stimulus interval.
Exposure 2: 916 kHz FUS (continuous and pulsed) to visual cortex (near Lambda)
Target: visual cortex — “0.5 mm anterior and 2.5 mm lateral of Lambda (visual cortex)”
Device: Sonic Concepts · Sonic Concepts · H-115 ✓
| Waveform | continuous, pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 916 | ✓✓✓ |
| Pulse duration (ms) | 0.229, 0.2, 20, 2swept | ✓✓✓ |
| Pulse repetition frequency (Hz) | 2,500, 1,500, 30, 300swept | ✓✓✓ |
| Duty cycle (%) | 100, 57.3, 30, 60swept | ✓? |
| Sonication duration (s) | 0.5, 0.06, 0.08, 0.067swept | ✓✓✓ |
| Free-field pressure (kPa) | 100, 2,400swept | ✓✓✓⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | 0.33, 5.33, 16.33, 33.33swept | ✓✓✓⚑ |
| Free-field Ispta (W/cm²) | 0.189, 1.72, 0.099, 0.9, 0.105, 1.8swept | ✓✓✓ |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
916 kHz (third harmonic) FUS at the same visual-cortex target as the 270 kHz exposure. Continuous-wave stimulation used a 500 ms pulse duration and was swept from 100 kPa up to 2,400 kPa across clear-skull and TPX cranial-window preparations (specific tested values included 100, 300, 400, 500, 700, 900, 1000, 1300, 1600, 2000 and 2400 kPa in different figures/sub-experiments), with a 20 s inter-stimulus interval for most experiments (155 s for the high-pressure long-ISI experiment in Figure S3) and 50 blocks per animal. Separately, eight pulsed ultrasound parameter sets (Table 1, 'Set1' replicating Tufail et al. and 'Set2' replicating Yu et al. pulse patterns) were also tested at 916 kHz at 100 kPa and 300 kPa, with pulse duration/PRF/duty cycle/number of pulses (Np) as tabulated, producing burst (sonication) lengths of 60, 80 or 67 ms.
Flags from extraction
exposures[1]— 916 kHz continuous-wave sweep and Table 1 pulsed parameter sets are combined into one exposure per the target x frequency rule, even though waveform differs; this makes several timing list fields heterogeneous (mixing continuous and pulsed conditions).exposures[1].free_field.pressure_kpa— Recorded as the paper's stated overall range (100-2400 kPa) rather than every discrete pressure value tested across different figures/sub-experiments (100,300,400,500,700,900,1000,1300,1600,2000,2400 kPa); individual values are in protocol_description.exposures[1].free_field.isppa_w_cm2— The four intensity values quoted (Figure 4D) correspond only to the TPX-window continuous 100/400/700/1000 kPa subset, not the full 100-2400 kPa pressure range or the pulsed Table 1 conditions (which report separate ISPPA/ISPTA values in mW/cm2 not fully reproduced here).exposures[0].timing.sonication_duration_s— Value (0.5 s) inferred from the stated 500 ms pulse duration (PD) used for all continuous-wave experiments at both frequencies; the anchor quote does not itself state the duration number.n_subjects— Different figures/panels report different animal numbers (e.g., n=6, n=4, n=3, n=10) across overlapping sub-experiments and preparations (clear skull vs TPX window, deafened vs normal hearing); no single total number of ultrasound-exposed animals is stated.direction_of_effect— Overall classified as no_effect for direct cortical neuromodulation, but very high pressures (>=1600 kPa) produced strong focal calcium activation attributed to tissue damage/spreading depolarization rather than physiological neuromodulation; see direction_notes.